Phytase mutants

ABSTRACT

Provided are mutants PHY1, PHY4 and PHY5 of a wild-type phytase APPA. After being treated for 10 min at 80° C., the residual enzyme activities of the mutants PHY1, PHY4 and PHY5 are respectively higher by 33.85%, 53.11% and 75.86% compared with that of APPA-M; after being treated for 5 min at 85° C., the residual enzyme activities of the mutants PHY1, PHY4 and PHY5 are respectively higher by 14.89%, 28.45% and 44.94% compared with that of APPA-M, and the heat resistance of these mutants is significantly higher than that of APPA-M.

This application claims priority to Chinese application No.201410677220.8, named “Phytase mutants”, filed on Nov. 21, 2014, thecontents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to biotechnology field, and particularlyrelates to phytase mutants, the method for producing the mutants and theuses thereof. The present invention also relates to DNA moleculesencoding the mutants, expression vectors, and host cells.

Reference to Submission of a Sequence Listing as a Text File

The Sequence Listing written in file2014-12-08_101788-1043152-000100US.txt created on Oct. 12, 2018, 27,130bytes, machine format IBM-PC, MS-Windows operating system, is herebyincorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Phytase is a type of phosphatase enzyme and can hydrolyze phytatephosphorus (myo-inositol hexakisphosphate) into myo-inositol andinorganic phosphate. There are two types of phytase: 3-phytase (EC3.1.3.8) and 6-phytase (EC 3.1.2.6). Phytase is widely spread in nature,occurring in plants, animals and microorganisms, including higher plantssuch as maize and wheat, prokaryotic microbes such as Bacillus subtilis,Pseudomonas, Lactobacillus and Escherichia coli, eukaryotic microbessuch as yeast, Rhizopus and Aspergillus.

Phytate phosphorus is a major component of all plant seeds, constituting1%-3% by weight of many cereals, beans and oilseeds and typicallyaccounting for 60%-80% of the total phosphorus. However, mono gastricanimals metabolize only 0%-40% of the phytate phosphorus since they lackdigestive enzymes for phytate, which results in a number of problems.First of all, phosphorus source are wasted. On one hand, phytatephosphorus source in feed cannot be efficiently utilized, on the otherhand, in order to ensure that the animals' requirement for phosphorus,it is necessary to add inorganic phosphorus in feed, which increases thefeed costs. Secondly, the excreta with high phosphorus pollute theenvironment. 85% of the phytate phosphorus in feed will be directlyexcreted by animals, and the excreta containing high phytate phosphoruswill pollute the water and soil seriously. In addition, phytatephosphorus is also a kind of antinutrient, which binds to severalmetallic ions such as Zn²⁺, Ca²⁺, Cu²⁺ and Fe²⁺ and other proteins toform insoluble compositions, preventing or inhibiting the absorption ofthe nutrients in the gastrointestinal tract, and reduces the effectiveutilization of nutrients.

Phytase can be used as a feed additive for mono gastric animals, and thefeeding effect has been worldwide confirmed. Phytase can improve thephosphorus availability of plant feeds by 60% and decrease thephosphorus excretion by 40%. Phytase also can counteract theanti-nutritional properties of phytate. Therefore, the addition ofphytase in animal feed is helpful for improving the productionefficiency of livestock and poultry industry and for reducing theenvironmental pollution caused by phytate.

There are two main kinds of phytase for industrial production, one ofwhich is fungal phytase derived from Aspergillus niger and the other isbacterial phytase derived from E. coli. The phytase APPA derived from E.coli has high specific activity and good gastrointestinal stability, andcan be used in the feed industry by addition to mash feed directly orspraying on pelleted feed.

Bacterial phytase APPA has lower heat stability, the retention rate ofwhich was even less than 30% after 70 degree Celsius (° C.) for 5minutes in water bath. Thus there is is a restriction of adding phytasedirectly into feed processing due to its low resistance on hightemperature of 80-90° C. in feed pelleting period. However, there arestill several disadvantages of applying liquid spraying technology usingphytase, such as high equipment cost, less stability and uniformity ofenzymes in the feed. Therefore it is of great importance to improvethermostability of phytase for feed.

SUMMARY OF THE INVENTION

This invention provides phytase mutants and the production methodsthereof. The phytase mutants have enhanced thermostabilities, which isconducive to the wide applications of the phytase mutants in the feedfield.

This invention provides phytase mutants comprising the amino acidsequences shown in (I) and (II):

(I) an amino acid sequence which has at least 70% identity to the aminoacid sequence of the wild-type phytase;

(II) an amino acid sequence which has at least one immune epitope of thephytase, and comprises a modification, substitution, deletion, and/orinsertion of one or more amino acids within the amino acid sequence ofthe wild-type phytase.

In some embodiments of the invention, the phytase mutants comprise aminoacid sequences which have at least 75% identity to the amino acidsequence of the wild-type phytase.

In other embodiments, the phytase mutants comprise amino acid sequenceswhich have at least 80% identity to the amino acid sequence of thewild-type phytase.

In other embodiments, the phytase mutants comprise amino acid sequenceswhich have at least 85% identity to the amino acid sequence of thewild-type phytase.

In other embodiments, the phytase mutants comprise amino acid sequenceswhich have at least 90% identity to the amino acid sequence of thewild-type phytase.

In other embodiments, the phytase mutants comprise amino acid sequenceswhich have at least 95% identity to the amino acid sequence of thewild-type phytase.

In some embodiments, the modifications include amidation,phosphorylation, methylation, acetylation, ubiquitination,glycosylation, or carbonylation.

In other embodiments, the phytase mutants comprise 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acid substitutions withinthe amino acid sequence encoding the phytase.

In some embodiments of the invention, the phytase mutants comprise 12,13, 16 or 17 amino acid substitutions within the amino acid sequence ofthe wild-type phytase.

In other embodiments, the phytase mutants have one or more amino acidsubstitutions in a position selected from position 25, 46, 62, 70, 73,75, 114, 137, 142, 146, 159 and 255, the positions corresponding to therespective position in the amino acid sequence of the wild-type phytase.

In some embodiments of the invention, the amino acid sequence of thewild-type phytase is SEQ ID NO: 1.

In further embodiments, the phytase mutants comprise 12 amino acidsubstitutions, wherein the amino acid substitutions are in positions 25,46, 62, 70, 73, 75, 114, 137, 142, 146, 159 and 255, and thesubstitutions are 25F, 46E, 62W, 70E, 73P, 75C, 114H, 137V, 142R, 146E,159Y and 255D, the position corresponding to the respective position inSEQ ID NO: 1.

The invention also provides DNA molecules comprising a polynucleotidesequence encoding a phytase mutant described herein.

In other embodiments, the phytase mutants have amino acid sequences ofSEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9.

In other embodiments, the DNA molecules encoding phytase mutants havepolynucleotide sequences of SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8or SEQ ID NO: 10.

The invention also provides vectors containing the DNA moleculesencoding phytase mutants.

In other embodiment, the phytase mutant has the amino acid sequence ofSEQ ID NO: 3, and one of the polynucleotide sequences encoding the sameis SEQ ID NO: 4.

The invention also provides the plasmid containing the polynucleotidesequence of SEQ ID NO: 4.

In other embodiments, the phytase mutants also comprise amino acidsubstitutions in position 380, the position corresponding to therespective position in SEQ ID NO: 1.

In other embodiments, the amino acid substitution in position 380 is380P (from Ala to Pro).

In other embodiment, the phytase mutant has the amino acid sequence ofSEQ ID NO: 5, and one of the polynucleotide sequences encoding the sameis SEQ ID NO: 6.

The invention also provides plasmids containing the polynucleotidesequence of SEQ ID NO: 6.

In other embodiments, the phytase mutants also comprise one or moreamino acid substitutions in position 80, 176 and 187, the positioncorresponding to the respective position in SEQ ID NO: 1.

In other embodiments, the amino acid substitution in position 80 is 80P(from Ser to Pro), the amino acid substitution in position 176 is 176P(from Asn to Pro), the amino acid substitution in position 187 is 187P(from Ser to Pro).

In other embodiment, the phytase mutant has the amino acid sequence ofSEQ ID NO: 7, and one of the polynucleotide sequences encoding the sameis SEQ ID NO: 8.

The invention also provides plasmids containing the polynucleotidesequence of SEQ ID NO:8.

In other embodiments, the phytase mutants also comprise the amino acidsubstitutions in position 161, the position corresponding to therespective position in SEQ ID NO: 1.

In other embodiments, the amino acid substitution in position 161 is161P (from Thr to Pro).

In other embodiment, the phytase mutant has the amino acid sequence ofSEQ ID NO: 9, and one of the polynucleotide sequences encoding the sameis SEQ ID NO: 10.

The invention also provides plasmids containing the nucleic acidsequence of SEQ ID NO:10.

The invention also provides the methods of producing the phytasemutants, which include:

Step 1: obtain a DNA molecule comprising a polynucleotide sequenceencoding any one of the amino acid sequences shown in (I) and (II):

(I) an amino acid sequence which has at least 70% identity to the aminoacid sequence of a wild-type phytase;

(II) an amino acid sequence which has at least one immune epitope of thephytase, and comprise a modification, substitution, deletion, and/orinsertion of one or more amino acids of the amino acid sequence of thephytase.

Step 2: fuse the DNA molecule obtained by step 1 to the expressionvectors, construct recombinant expression vectors, and transform therecombinant expression vectors into the host cells;

Step 3: induce the host cells containing recombinant expression vectorsto express the fusion protein, and then isolate and purify the fusionprotein.

In some embodiments of the invention, the modifications in the method ofproducing the phytase mutants include amidation, phosphorylation,methylation, acetylation, ubiquitination, glycosylation, orcarbonylation.

In other embodiments, the substitutions in the method of producing thephytase mutants include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 or 17 amino acid substitutions within the amino acid sequence ofthe phytase.

In other embodiments, the substitutions in the method of producing thephytase mutants include one or more amino acid substitutions in aposition selected from position 25, 46, 62, 70, 73, 75, 114, 137, 142,146, 159 and 255, the positions corresponding to the respective positionin the amino acid sequence of the phytase.

In other embodiments, the substitutions in the method of producing thephytase mutants also include amino acid substitutions in position 380,the positions corresponding to the respective position in the amino acidsequence encoding the phytase.

In other embodiments, the substitutions in the method of producing thephytase mutants further include one or more amino acid substitutions inposition 80, 176 and 187, the positions corresponding to the respectiveposition in the amino acid sequence of the phytase.

In other embodiments, the substitutions in the method of producing thephytase mutants further include amino acid substitutions in position161, the positions corresponding to the respective position in the aminoacid sequence of the phytase.

In some embodiments, the DNA molecules in step 1 of the method areobtained by amplification reactions of cDNA encoding the amino acidsequence of SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:9.

The host cell in Step 2 of the method is Pichia.

The invention also provides a modified feed such as feed for monogastric animals comprising an effective amount of a phytase mutantdescribed herein.

The invention also provides host cells containing the recombinantexpression vectors.

In some embodiments, the host cell is Pichia.

The recombinant phytase mutants expressed in the Pichia containing theplasmid are more heat-resistant.

The invention provides phytase mutants comprising any one of the aminoacid sequences shown in (I) and (II):

(I) an amino acid sequence which has at least 70% identity to the aminoacid sequence of a wild-type phytase;

(II) an amino acid sequence which has at least one immune epitope of thephytase, and comprise a modification, substitution, deletion, and/orinsertion of one or more amino acids of the amino acid sequence of thephytase.

There are four phytase mutants provided in the invention with improvedheat resistance. After being treated at 75° C. for 5 minutes, theresidual enzyme activity of the mutant APPA-M was higher than 95%, whilethat of the wild-type phytase APPA was lower than 10%. Using the mutantAPPA-M as a basis, the invention also provided an additional one-pointmutant PHY1 (A380P), an additional four-point mutant PHY4 (S80P, T161P,N176P and A380P) and an additional five-point mutant PHY5 (S80P, T161P,N176P, S187P and A380P). After being treated at 80° C. for 10 min, theresidual enzyme activities of the mutants PHY1, PHY4 and PHY5 werehigher by 33.85%, 53.11% and 75.86% respectively compared with that ofAPPA-M. After being treated at 85° C. for 5 min, the residual enzymeactivities of the mutants PHY1, PHY4 and PHY5 were higher by 14.89%,28.45% and 44.94% respectively compared with that of APPA-M. The heatresistance of these mutants are significantly higher than that ofAPPA-M, which will improve the applications of the phytase mutants infeed.

BRIEF DESCRIPTIONS OF DRAWINGS

FIG. 1 shows the map of recombinant plasmid pPIC9K-APPA;

FIG. 2 shows the thermostability of PHY1, PHY4 and PHY5 compared withthat of APPA-M.

EMBODIMENT

The invention discloses phytase mutants, methods of production and theuses thereof, DNA molecules encoding the mutants, vectors, and hostcells. Technicians having ordinary skill in the field can learn from thecontents of this invention and improve the process parameters to realizeit. It is particularly to be noted that all similar substitutions andmodifications will be regarded as obvious and are considered to beincluded in the invention. The invention has described the methods andapplications in the preferred embodiments, and technicians in this fieldcan readily modify or appropriately modify and combine the methods andapplications to realize and apply the invention without departing fromthe contents, spirit and scope of the invention.

Conventional techniques and methods in the field of genetic engineeringand molecular biology are used in the invention, for example, themethods recorded in MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed.(Sambrook, 2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel,2003). These general references provide one of skill with a generaldictionary of many of the terms used in this invention. Based on thetechnical scheme described in the invention, all technical andscientific terms can choose other conventional methods, experimentalprograms and reagents to realize the invention, not limited to thatdescribed in the embodiments of the invention. For example, thefollowing experimental materials and reagents can be used in theinvention:

Strains and vectors: E. coli DH5α, Pichia pastoris strain GS115, vectorpPIC9k were purchased from Invitrogen.

Reagents: Amp and G418 were purchased from Invitrogen.

Enzymes and Kits: PCR enzymes and ligases were purchased from Takara;restriction endonucleases were purchased from Fermentas; plasmid minikit and gel extraction kit were purchased from Omega; geneMorph IIrandom mutagenesis kit was purchased from MBL Beijing Biotech Co., Ltd.

Medium Recipes:

Lariant broth (LB medium): 0.5% yeast extract, 1% tryptone, 1% NaCl,pH7.0;

LB-AMP medium: LB medium with 100 μg/mL ampicillin;

Yeast extract peptone dextrose medium (YPD medium): 1% yeast extract, 1%tryptone, 1% glucose;

Minimal dextrose medium (MD medium): 2% tryptone, 2% agar;

BMGY medium: 2% tryptone, 1% yeast extract, 100 mM potassium phosphatebuffer (pH 6.0), 1.34% YNB, 4×10⁻⁵ biotin, 1% glycerol;

BMMY medium: 2% tryptone, 1% yeast extract, 100 mM potassium phosphatebuffer (pH 6.0), 1.34% YNB, 4×10⁻⁵ biotin, 1% methanol.

The invention was further illustrated by the following examples:

Example 1 Phytase Mutants

Gene synthesis of the wild-type phytase APPA and phytase mutant APPA-MThe wild-type phytase APPA was derived from E. coli, of which the aminoacid sequence was SEQ ID NO:1 and the encoding polynucleotide sequencewas SEQ ID NO: 2. In order to improve the thermostability of APPA, aphytase mutant was obtained by introducing 12 point-mutations into theamino acid sequence of SEQ ID NO:1, which were A25F, W46E, Q62W, G70E,A73P, K75C, T114H, N137V, D142R, S146E, R159Y, Y255D.

The phytase mutant was named APPA-M, of which the amino acid sequencewas SEQ ID NO: 3 and the encoding polynucleotide sequence was SEQ ID NO:4. The polynucleotide sequence was optimized according to the codonpreference of Pichia pastoris and synthesized by Shanghai GenerayBiotech Co., Ltd with an EcoRI restriction site and a NotI restrictionsite added to the 5′ end and 3′ end respectively.

The same method above was used to synthesize the polynucleotide sequenceof the wild-type phytase APPA.

Construction of the Expression Vector Carrying Phytase Gene

The two polynucleotide sequences synthesized in example 1.1 and theplasmid pPIC-9k were first digested by EcoRI and NotI, and then ligatedtogether at 16° C. overnight respectively. After that, the recombinantplasmid was transformed into E. coli DH5α. The recombinant E. colistrains then were spread onto LB+Amp plates. The plates were placedinverted and incubated at 37° C. until transformants grew up. Positivetransfromants were selected and verified by colony PCR and DNAsequencing, and named as pPIC9K-APPA (the map of pPIC9K-APPA were shownin FIG. 1) and pPIC9K-APPA-M respectively. The reaction system of colonyPCR contained: monoclonal sample, rTaqDNA polymerase 0.5 ul, 10×Buffer2.0 μL, dNTPs (2.5 mM) 2.0 μL, 5′AOX primer (10M) 0.5 μL, 3′AOX primer0.5 μL, ddH2O 14.5 μL; PCR conditions were: 95° C. for 5 min (1 cycle),94° C. for 30 sec, 55° C. for 30 sec, 72° C. for 2 min (30 cycles), and72° C. for 10 min (1 cycle).

Construction of the Recombinant P. pastoris Strains

Preparation of Competent P. pastoris Cells

Host cells R pastoris GS115 were spread onto YPD plates and the plateswere incubated at 30° C. for 48 h. GS115 colonies were picked up andinoculated into 6 mL YPD liquid medium and incubated for approximately12 h at 30° C. with shaking at 220 rpm. Then the YPD liquid mediumcontaining GS115 was inoculated into 30 mL YPD liquid medium andincubated for 5 h at 30° C. with shaking at 220 rpm. The cell density ofthe yeast cultures were measured using a spectrophotometer. When theoptical density (OD600) between 1.1 and 1.3, 4 mL yeast cultures wereadded into a sterilized EP tubes and centrifuged at 9000 rpm and 4° C.for 2 min. The supernatants were removed, while the remaining yeastcells were re-suspended in 1 ml of sterile pre-cooled water. Thesuspension containing yeast cells was centrifuged at 9000 rpm and 4° C.for 2 min. The supernatant was removed, while the remaining yeast cellswere re-suspended in 1 ml of pre-cooled sorbitol (1 mol/L). The sorbitolcontaining yeast cells was centrifuged at 9000 rpm and 4° C. for 2 min.The supernatant was removed, while the remaining yeast cells werere-suspended in 100-150 μl of sterile pre-cooled sorbitol (1 mol/L).

1.3.2 Transformation and Screening

The recombinant plasmids pPIC9K-APPA and pPIC9K-APPA-M were linearizedby Sal I and transformed into Pichia pastoris GS115 respectively byelectroporation. Then the transformation mixtures were spread on MDplates and dried in a sterile bench. The MD plates were placed invertedand incubated at 30° C. for 2-3 days to obtain recombinant P. pastorisstrains carrying the recombinant plasmids pPIC9K-APPA or pPIC9K-APPA-M.There were approximately 300 clones on each plate. The clones werewashed down with sterile water and spread on YPD plates containingdifferent concentrations of geneticin (0.5 mg/mL-8 mg/mL) to screenmultiple copies of transformants.

One of the recombinant yeast strains carrying the recombinant plasmidspPIC9K-APPA was named Pichia pastoris APPA. One of the recombinant yeaststrains carrying the recombinant plasmids pPIC9K-APPA-M was named Pichiapastoris APPA-M. The two recombinant strains were first inoculated intoseparate flasks with BMGY medium and cultured at 30° C. for 1 d withagitation at 250 rpm, and then inoculated in BMMY medium at 30° C. for 4d with agitation at 250 rpm. 0.5% methanol was added into the medium asan inducer every day. After that, the medium was centrifuged at 9000 rpmfor 10 min. The fermentation supernatants containing phytase wereretained, while the yeast cells were removed.

(1) Definition of Phytase Activity Unit

One phytase unit is the activity of phytase that generates 1 micromoleof inorganic phosphorus per minute from 5.0 mmol/L sodium phytate at pH5.0 and 37° C., which is indicated as U.

Method for Detecting Phytase Activity

1.8 mL of acetic acid buffer (pH 5.0) and 0.2 mL of sample are bothadded into two separate cuvettes named A and B, mixed and warmed at 37°C. for 5 min. 4 mL of substrate solution is added into cuvette A and 4mL of stop solution is added into cuvette B, mixed and reacted at 37° C.for 30 min. The reaction is ended by adding and mixing 4 mL stopsolution in cuvette A and 4 mL substrate solution in cuvette B. Afterstanding for 3 min, the absorbance is measured at 415 nm. Three repeatsare made for each sample, and the average of the absorbance values isused for calculating the phytase activity by regression linear.X=F×C/(m×30)  Enzyme activity:

where: X—Unit of enzyme activity, U/g(mL);

F—Total dilution factors of sample solution before reaction;

C—The enzyme activity is calculated from the linear regression equationbased on the absorbance of the actual sample solution, U;

m—Sample mass or volume, g/mL; Reaction time;

30—Reaction time;

(3) Phytase Activities were Shown in Table 1

TABLE 1 Phytase activities Activity Sample Value 1 Value 2 Value 3Average (U/mL) APPA 0.473 0.477 0.471 0.474 166 APPA-M 0.486 0.489 0.4840.486 195

As shown in Table 1, the enzyme activities of the fermentationsupernatants of Pichia pastoris APPA and Pichia pastoris APPA-M were 166U/mL and 195 U/mL, respectively.

Fermentation Process

P. pastoris APPA and P. pastoris APPA-M were cultured in two separate 10L fermenters with the fermentation medium containing: 1.1 g/L CaSO₄, 5.5g/L KH₂PO₄, 55 g/L NH₄H₂PO₄, 16.4 g/L MgSO₄, 20.3 g/L K₂SO₄, 1.65 g/LKOH and 0.05% antifoam, and the fermentation parameters: pH 5.0, 30° C.,agitation at 300 rpm, aeration at 1.0-1.5 v/v, and the dissolved oxygenkept above 20%.

There were three stages of the fermentation process. The first stage wasfor cell culture with 7% seed inoculated and cultured at 30° C. for24-26 h until the supplement of glucose was finished. The second stagewas for cell hunger with no more carbon source supplemented. This stagelasted about 30-60 min until the concentration of dissolved oxygen roseto 80%. The third stage was for inducing the expression of phytase withmethanol added as an inducer in flow, and the concentration of dissolvedoxygen maintained at more than 20%, which lasted about 150-180 h. Afterthat, the fermentation broth was treated by a plate and frame filter toobtain crude enzyme solution.

The phytase activities of the crude enzyme solutions were determined bythe method mentioned in 1.3.2, and the results were shown in Table 2.

TABLE 2 Phytase activity test results Activity Sample Value 1 Value 2Value 3 Average (U/mL) APPA 0.488 0.485 0.487 0.487 9800 APPA-M 0.4590.461 0.462 0.461 10257

The phytase activities of the crude enzyme solutions of P. pastoris APPAand P. pastoris APPA-M were 9800 U/mL and 10257 U/mL, respectively.

Analysis of Enzymatic Properties

Optimal Temperature

The phytase activities of the crude enzyme solutions of P. pastoris APPAand P. pastoris APPA-M were measured at pH5.5 and 5° C. intervalsbetween 30° C. and 85° C. With the highest phytase activity calculated100%, the relative enzyme activities were calculated. The results showedthat the optimal temperatures of the wild-type phytase APPA and phytasemutant APPA-M were both 75° C.

Optimal pH

The crude enzyme solutions of P. pastoris APPA and P. pastoris APPA-Mwere diluted by 0.1M acetic acid-sodium acetate buffer at pH 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 respectively. The phytaseactivities were measured at 37° C., and the relative enzyme activitieswere calculated with the highest enzyme activity calculated 100%. Theresults showed that the optimal pH of wild phytase APPA and phytasemutant APPA-M was both 5.0.

Thermostability

The crude enzyme solutions of P. pastoris APPA and P. pastoris APPA-Mwere diluted 10 times with 0.25M sodium acetate buffer (pH 5.0) whichwas preheated for 10 min. The diluted enzyme solutions were well mixedand treated at 75° C. for 5 min.

The phytase activities were measured when the diluted enzyme solutionswere cooled to room temperature. With the phytase activity of theuntreated enzyme solution calculated 100%, the residual phytaseactivities were calculated.Residual phytase activity (%)=phytase activity of the enzyme solutionbeing treated/phytase activity of the enzyme solution beinguntreated×100%.

The results showed that after being treated at 75° C. for 5 min, theresidual phytase activity of the wild-type phytase APPA was below 10%,while that of the phytase mutant APPA-M was above 95%. In conclusion,the thermostability of the phytase mutant APPA-M was significantlyhigher than that of the wild-type phytase APPA.

Example 2 Phytase Mutants

In order to improve the thermostability of the phytase mutant APPA-M,the protein structure of APPA-M was analyzed. The result showed thatthere were two domains in the protein: domain I contained 134 amino acidresidues at the N-terminus and 152 amino acid residues at C-terminus,while domain II contained the remaining 124 amino acid residues in themiddle. The conserved sequences and activity center are all in domain I.Without destroying the secondary structure and activity center of theprotein, Further mutations of the amino acid residuals were carried out.

2.1 Mutations of Phytase Mutant APPA-M

Primer APPAM-FI and APPAM-R1 were designed: XynII-F1:GGCGAATTCCAGTCAGAACCAGAGTTGAAGTT (Underlined was the recognition site of restriction endonuclease EcoRI),which was shown in SEQ ID NO: 11; XynII-R1:ATAGCGGCCGCTTACAAGGAACAAGCAGGGAT (Underlined was the recognition site of restriction endonuclease Notl),which was shown in SEQ ID NO: 12;

APPA-M gene was amplified using the primers above by a GeneMorph IIrandom mutagenesis kit. The amplification products were recovered, andthen digested with EcoRI and NotI and ligated into EcoRI-NotI-digestedplasmid pET21a. After that the plasmid was transformed into E. coli BL21(DE3) and then the recombinant E. coli cells were spread onto LB+Ampplates. After being incubated at 37° C., the colonies were transferredone by one into 96-well polypropylene microtiter plates containingLB+Amp medium with 150 ul 0.1 mM IPTG in each well. The microtiterplates were incubated at 37° C. for 6 h with shaking at 220 rpm. Thesupernatant was removed from the fermentation broth by centrifugation.Afterwards the cells were re-suspended with buffer and repeatedfreeze-thawed to obtain phytase-containing E. coli cell lysates.

40 ul cell lysates were transferred into two separate new 96-wellplates, one of which was treated at 80° C. for 10 min, and the other wasnot. 80 ul substrates were added into each well of the plates andincubated for 30 min at 37° C. Afterwards 80 ul stop solution (ammoniumvanadate:ammonium molybdate:nitric acid=1:1:2) was added to end thereaction. In each well of the plates, the contents of inorganicphosphate were determined, which reflected the activities of differentmutants obtained in the invention.

Compared with phytase APPA-M, the thermostabilities of some mutants werenot improved, or even worse. For example, after being treated at 80° C.for 5 min, the residual enzyme activities of a three-point mutant(Q184E/Y289K/1405L) and the C-terminal (CNZSMQTD) removed mutant werereduced by 9% and 17% respectively, and two one-point mutants (Q285Y andC178N) were almost inactivated. Besides, there were some mutants withimproved thermostabilities, but their enzymatic properties weresignificantly changed, which also limited their applications in feed.

This invention provided three mutants with significantly improvedthermostabilities as well as high activities and original enzymaticproperties.

One mutant was named PHY1 with one-point mutation A380P, its amino acidsequence was shown as SEQ ID NO: 5, and the encoding polynucleotidesequence was shown as SEQ ID NO: 6.

Another mutant was named PHY4 with four-point mutations S80P, N176P,S187P and A380P, its amino acid sequence was shown as SEQ ID NO: 7, andthe encoding polynucleotide sequence was shown as SEQ ID NO: 8.

The other mutant was named PHY5 with five-point mutations S80P, T161P,N176P, S187P and A380P, its amino acid sequence was shown as SEQ ID NO:9, and the encoding polynucleotide sequence was shown as SEQ ID NO: 10.

2.2 Synthesis and Amplification of Mutant Genes

Three polynucleotide sequences were synthesized with reference to SEQ IDNO: 6, SEQ ID NO: 8 and SEQ ID NO: 10 and optimized based on codon biasof Pichia pastoris by Shanghai Generay Biotech Co., Ltd, of which anEcoRI restriction site and a NotI restriction site were added to the 5′end and 3′ end respectively.

2.3 Construction of Expression Vector

The three polynucleotide sequences synthesized above and the plasmidspPIC-9k were first digested by EcoRI and NotI, and then ligated togetherat 16° C. overnight respectively. After that, the recombinant plasmidwas transformed into E. coli DH5α. The recombinant E. coli cells thenwere spread onto LB+Amp plates. The plates were placed inverted andincubated at 37° C. until transformants grew up. Positive transfromantswere selected and verified by colony PCR (reaction was as same as inExample 1) and DNA sequencing, and were named as pPIC9K-PHY1,pPIC9K-PHY4 and pPIC9K-PHY5 respectively.

2.4 Construction of the Recombinant P. pastoris Strain

The recombinant plasmids pPIC9K-PHY1, pPIC9K-PHY4 and pPIC9K-PHY5 werelinearized by Sal I and transformed into host cells Pichia pastorisGS115 by electroporation. The recombinant strains P. pastorisGS115/pPIC9K-PHY1, GS115/pPIC9K-PHY4 and GS115/pPIC9K-PHY5 were obtainedon MD plates after screening YPD plates containing differentconcentrations of geneticin (0.5 mg/mL-8 mg/mL) were used to selectmultiple copies of transformants.

The transformants of the recombinant strains GS115/pPIC9K-PHY1,GS115/pPIC9K-PHY4 and GS115/pPIC9K-PHY5 were named Pichia pastoris PHY1,Pichia pastoris PHY4, and Pichia pastoris PHY5, respectively. The threetransformants above were inoculated into separate flasks with BMGYmedium and cultured at 30° C. for 1 d with agitation at 250 rpm, andthen transferred and inoculated in BMMY medium at 30° C. for 4 d withagitation at 250 rpm. 0.5% methanol, as an inducer, was added every 24h. The cells were removed from the fermentation broth by centrifugationat 9000 rpm for 10 min and the fermentation supernatants containingphytase PHY1, or phytase PHY4 or phytase PHY5 were retained.

The activities of fermentation supernatants were detected by the methodmentioned in 1.3.2, and the results were shown in Table 3.

TABLE 3 Phytase activities Activity Sample Value 1 Value 2 Value 3Average (U/mL) PHY1 0.481 0.483 0.484 0.482 211 PHY4 0.483 0.479 0.4810.481 201 PHY5 0.491 0.488 0.489 0.489 255

As shown in Table 3, the activities of the fermentation supernatants ofPichia pastoris PHY1, PHY4 and PHY5 were 211 U/mL, 201 U/mL and 255U/mL, respectively.

2.5 Fermentation Process P. pastoris PHY1, P. pastoris PHY4 and P.pastoris PHY5 were fermented in three separate 10 L fermenters. Thefermentation medium contained 1.1 g/L CaSO₄, 5.5 g/L KH₂PO₄, 55 g/LNH₄H₂PO₄, 16.4 g/L MgSO₄, 20.3 g/L K₂SO₄, 1.65 g/L KOH and 0.05%antifoam.

Fermentation parameters: pH 5.0, 30° C., agitation at 300 rpm, aerationat 1.0-1.5 v/v, and the dissolved oxygen was kept above 20%.

There were three stages of the fermentation process. The first stage wasfor cell culture with 7% seed inoculated and cultured at 30° C. for24-26 h until the supplement of glucose was finished. The second stagewas for cell hunger with no more carbon source supplemented. This stagelasted about 30-60 min until the concentration of dissolved oxygen roseto 80%. The third stage was for inducing the expression of phytase withmethanol added as an inducer in flow, and the concentration of dissolvedoxygen maintained at more than 20%, which lasted about 150-180 h. Afterthat, the fermentation broth was treated by a plate and frame filter toobtain crude enzyme solution.

The phytase activities of the crude enzyme solutions were detected bythe method mentioned in 1.3.2, and the results were shown in Table 4.

TABLE 4 Phytase activities Activity Sample Value 1 Value 2 Value 3Average (U/mL) PHY1 0.478 0.479 0.481 0.479 10317 PHY4 0.484 0.480 0.4810.482 10401 PHY5 0.479 0.477 0.480 0.479 10813

The phytase activities of the crude enzyme solutions of R pastoris PHY1,P. pastoris PHY4 and P. pastoris PHY5 were 10317 U/mL, 10401 U/mL, and10813 U/mL, respectively.

Analysis of Enzymatic Properties

Optimal Temperature

The phytase activities of the crude enzyme solutions of P. pastorisPHY1, PHY4 and PHY5 were measured at pH5.5 and 5° C. intervals between30° C. and 85° C. With the highest phytase activity calculated 100%, therelative enzyme activities were calculated. The results showed that theoptimal temperatures of phytase mutants PHY1, PHY4 and PHY5 were 75° C.,which were the same with the wild-type phytase APPA and the mutantAPPA-M.

Optimal pH

The crude enzyme solutions of P. pastoris PHY1, PHY4 and PHY5 werediluted by 0.1M acetic acid-sodium acetate buffer at pH 2.0, 2.5, 3.0,3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 respectively. The phytaseactivities were measured at 37° C., and the relative enzyme activitieswere calculated with the highest enzyme activity calculated 100%. Theresults showed that the optimal pH of the phytase mutants PHY1, PHY4 andPHY5 were 5.0, which were the same with the wild-type phytase APPA andthe mutant APPA-M.

Thermostability

The crude enzyme solutions of P. pastoris PHY1, PHY4 and PHY5 werediluted 10 times with 0.25M sodium acetate buffer (pH 5.0) which waspreheated for 10 min. The diluted enzyme solutions were well mixed andtreated at 85° C. for 5 min, and 80° C. for 10 min, respectively. Thephytase activities were measured when the diluted enzyme solutions werecooled to room temperature. With the phytase activity of the untreatedenzyme solution calculated 100%, the residual phytase activities werecalculated.

As shown in FIG. 2, compared with phytase mutant APPA-M, the residualactivity of the phytase mutants PHY1, PHY4 and PHY5 were higher by33.85%, 53.11% and 75.86%, respectively, after being treated at 80° C.for 10 min, and were higher by 14.89%, 28.45% and 44.94%, respectively,after treating at 85° C. for 5 min.

In conclusion, Using the mutant APPA-M as a basis, the inventionprovided new mutants containing additional one- or multiple-pointmutations such as a one-point mutant PHY1 (A380P), a four-point mutantPHY4 (S80P, T161P, N176P and A380P) and a five-point mutant PHY5 (S80P,T161P, N176P, S187P and A380P). Compared with phytase mutant APPA-M, theoptimal pH of the phytase mutants PHY1, PHY4 and PHY5 remainedunchanged, but the thermostabilities of the phytase mutants PHY1, PHY4and PHY5 had been significantly increased, which was conducive to theapplications of the phytase mutants in feed.

The phytase mutants provided herein are described in detail. Theprinciples and embodiments of the invention have been described withreference to specific examples, and the descriptions of the aboveembodiments are merely illustrative of the method and the core idea ofthe present invention. It is particularly to be noted that all similarsubstitutions and modifications without departing from the principlewill be regarded as obvious to those skilled in the field and areconsidered to be fallen within the scope of the claims of the invention.

The invention claimed is:
 1. A phytase mutant comprising the amino acidsequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 orSEQ ID NO:
 9. 2. The phytase mutant of claim 1, consisting of the aminoacid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO:7 or SEQ ID NO:
 9. 3. A composition comprising the phytase mutant ofclaim 1 or
 2. 4. A feed for mono gastric animals, comprising aneffective amount of the phytase mutant of claim 1 or
 2. 5. A method forimproving a feed for mono gastric animals, comprising adding aneffective amount of the phytase mutant of claim 1 or 2 into the feed.